Compositions comprising bone marrow cells together with demineralized and/or mineralized bone matrix and uses thereof in the induction of bone and cartilage formation

ABSTRACT

A composition comprising bone marrow cells (BMC) and demineralized bone matrix (DBM) and/or mineralized bone matrix (MBM) and optionally comprising bone morphogenetic protein/s (BMP) and/or other active agents, particularly for use in the transplantation of mesenchymal progenitor cells into a joint and/or a cranio-facial-maxillary bone, for restoring and/or enhancing the formation of a new hyaline cartilage and subchondral bone structure. The composition of the invention and method of treatment employing the same may be used for the treatment of hereditary or acquired bone disorders, hereditary or acquired cartilage disorders, malignant bone or cartilage disorders, metabolic bone diseases, bone infections, conditions involving bone or cartilage deformities and Paget&#39;s disease. The composition and method may further be used for the correction of complex fractures, bone replacement and formation of new bone in plastic or sexual surgery, for support of implants of joints, cranio-facial-maxillary bones, or other musculoskeletal implants, including artificial implants. The method of the invention may further be used for treating damaged joints or degenerative arthropathy associated with malformation and/or dysfunction of cartilage and/or subchondral bone. A kit is provided for performing transplantation into a joint or a cranio-facial-maxillary bone of a mammal of the composition of the invention.

FIELD OF THE INVENTION

[0001] The present invention relates to compositions comprising bonemarrow cells (BMC) and demineralized and/or mineralized bone matrix (DBMand MBM, respectively) and to their novel uses in induction of new boneand cartilage formation in mammals.

BACKGROUND OF THE INVENTION

[0002] New bone formation, such as in the case of damage repair orsubstitution of a removed part of the bone in postnatal mammals, canonly occur in the presence of the following three essential components,(i) mesenchymal progenitor cells; (ii) a conductive scaffold for thesecells to infiltrate and populate; and (iii) Bone Morphogenetic Proteins.Unfortunately, local conditions usually do not satisfy the requirementsof osteogenesis, and thus substitution of removed, damaged or destroyedbones does not occur spontaneously.

[0003] Previous research has already uncovered somewhat about thesethree components.

[0004] It was shown that multipotent mesenchymal stem cells, which arecapable of extensive proliferation and differentiation into cartilage,bone, tendon, muscle, fat and etc. are present in the bone marrow[Caplan, A. I. (1991) J Orthop Res 9:641-650; Prockop J. D. (1997)Scieice 276:71-74; Pittehger, M. F. et al. (1999) Science 284:143-147;Wakitani, S. W. et al. (1995) Muscle & Nerve 18:1417-1426].

[0005] DBM (and/or MBM) has been shown to play the role of supportivematerial or structure that is essential for promoting engraftment ofmesenchymal progenitor cells and their proliferation and differentiationin the course of bone and cartilage development, whenever mesenchymalcells are introduced as a cell suspension (Inventor's unpublishedresults). It serves as a conductive scaffold for cartilage and boneregeneration, while providing a natural source for inducing bothchondro- and osteogenesis, thus combining all the essential inductiveand conductive features. DBM also has additional advantageous, that canbe summarized as follows: (i) it is mechanically flexible and slowlybiodegradable, with the degradation time compatible with the period ofde novo chondro- and osteogenesis; (ii) it is strong enough to provideat least partially biomechanical properties of the flat bone and jointsurface during the period of new bone and cartilage formation; (iii) itcan be provided as an amorphous powder that can be inserted locally,without major surgical intervention, while avoiding iatrogenic damage;(iv) it is a low immunogenic material even when used as a xenograft, andwhen used in an allogeneic combination, it is practicallynon-immunogenic [Block, J. E. and Poser, J. (1995) Med Hypotheses45(1):27-32; Torricelli, P. et al. (1999) Int Orthop 23(3):178-81;Hallfeldt, K. K. et al. (1995) J Surg Res 69(5):614-20].

[0006] BMPs are growth factors that play an important role in theformation of bone and cartilage [Ducy, P. and Karsenty, G. (2000) KidneyInt 57(6):2207-14; Schmitt, J. M. et al. (1999) J Orthop Res17(2):269-78]. Most importantly, DBM is a natural source of BMPs.Moreover, induction of cartilage and bone may be enhanced by additionalexogenous supply of BMPs that are not even species-specific [Sampath, T.K. and Reddi, A. H. (1983) Proc Natl Acad Sci USA 80(21): 6591-5;Bessho, K. et al. (1992) J Oral Maxillofac Surg 50(5):496-501], togetherwith DBM [Niederwanger, M. and Urist, M. R. (1996) J Oral Implantol22(3-4):210-5].

[0007] Arthropathies are a group of chronic progressive joint diseasesthat can result from degenerative changes in the cartilage andhypertrophy of bone at the articular margins. Arthropathies can besecondary to trauma, inflammatory (autoimmune or infectious), metabolicor neurogenic diseases. Hereditary and mechanical factors may be anadditional factor involved in the pathogenesis of arthropathies.

[0008] Restoration of a healthy joint surface in a damaged ordegenerative arthropathy requires addressing the treatment both towardsthe cartilage and the subchondral bone.

[0009] Various attempts have been made to replace damaged cartilage,including:

[0010] 1. Stimulation of bone marrow from subchondral bone to form afibrotic repair tissue;

[0011] 2. Osteochondral transplantation (allogeneic and autologous);

[0012] 3. Transplantation of autologous cultured chondrocyte ormesenchymal cells;

[0013] 4. Combined transplantation of chondrocytes with different kindsof matrices; and

[0014] 5. Artificial implantation of mechanical joints.

[0015] Each of these methods has limitations and disadvantages and mostof them are expensive, cumbersome, innefective and rather impractical.Autologous osteochondral graft is restricted to a small area of damagedcartilage, up to 2 cm², and could cause discomfort, infection andmorbidity in the donor site. Allogeneic osteochondral graft isimmunogenic, hence requires life-long use of undesired, hazardousimmunosuppressive agents, which would be an impractical approach forroutine orthopedic practice. Transplantation of cultured chondrocytes iscumbersome and very expensive, involving a two-stage procedure. Thehyaline-like tissue which is produced after transplantation hassub-optimal biomechanical properties [Gilbert, J. E. (1998) Am J KneeSurg 11(1):42-6; Temeno, J. S. and Mikos, A. G. (2000) Biomaterials:Tissue Engineering for Regeneration of Articular Cartilage, 21:431-440;Buckwalter, J. A. and Mankin, H. J. (1998) Instr Course Lect 47:487-504;Stocum, D. L. (1998) Wound Repair Regen. 6(4):276-90]. Hence, adequaterestoration of cartilage remains an unsolved problem.

[0016] Currently, autologous grafts are the most commonly used bone andcartilage graft material. However, the use of autografts haslimitations, such as donor site discomfort, infection and morbidity andlimited sizes and shapes of available grafts. Even if enough tissue istransplanted there is an acute limitation in the number of mesenchymalstem cells with high proliferative potential present in thedifferentiated bone tissue implanted.

[0017] In theory, the most promising approach should involve thecombined transplantation of cells capable of hyaline cartilage formationand a matrix, providing means for induction/conduction and support ofcartilage development and maintenance.

[0018] It is widely accepted that, for successful application ofcombined cell-matrix graft, the basic requirements are the following:

[0019] 1. Rich source of progenitor cells capable of differentiationinto chondrocytes, for continuous repair of “wear and tear” of weightbearing joints.

[0020] 2. Conductive scaffold for cell attachment should be maintained,leading to development of hyaline cartilage.

[0021] 3. Conductive scaffold should be non-immunogenic, non-toxic andsusceptible to biodegradation simultaneously with the development of newcartilage.

[0022] 4. Conditions for stimulating development of chondrocytes frommesenchymal precursor cells.

[0023] So far, most of the matrices that were tried in combinedcell-matrix grafts were either immunogenic or non-biodegradable, and theremaining others did not possess conductive or inductive propertiesneeded to support formation of biomechanical strong cartilage. Cellsused in combined cell-matrix grafts were in most of the caseschondrocytes, which were already fully differentiated cells, withrelatively low metabolic activity and limited self-renewal capacity.Whereas the proliferative capacity of such cells may be sufficient tomaintain healthy cartilage, it is certainly insufficient for thedevelopment de iLOVO of large areas of hyaline cartilage. In addition tobeing immunogenic, mesenchymal progenitor cell allografts were notcombined with optimal supportive matrix. Thus, unfortunately, none ofthe available options fulfill all basic requirements, and all optionsare far from being satisfactory for reliable routine clinicalapplication.

[0024] The composition of the invention comprising BMC and DBM and/orMBM overcomes the above shortcomings and provides, upon administrationinto a damaged joint, replacement and/or restoration of hyalinecartilage together with subchondral bone, in a one-step transplantationprocedure, without any preliminary cultivation of mesenchymal progenitorcells.

[0025] Thus, it is the major object of the present invention to providea mixture of bone marrow cells and demineralized or mineralized bonematrix, for use as a graft in patients in need of restoration of damagedjoints and cranio-facial-maxillary bones. This and other objects of theinvention will be elaborated on as the description proceeds.

SUMMARY OF THE INVENTION

[0026] The present invention relates to compositions comprising amixture of bone marrow cells (BMC) and demineralized and/or mineralizedbone matrix (DBM and MBM, respectively) and to their novel uses in thetransplantation of mesenchymal progenitor cells into joints andcranio-facial-maxillary bones.

[0027] Thus, in a first aspect, the present invention relates to acomposition comprising bone marrow cells (BMC) and demineralized bonematrix (DBM) and/or mineralized bone matrix (MBM).

[0028] In a second aspect, said composition comprising BMC and DBMand/or MBM is for use in transplantation of mesenchymal progenitor cellspresent in the bone marrow into a joint and/or a cranio-facial-maxillarybone of a subject in need, wherein said subject is a mammal, preferablya human.

[0029] In a first embodiment, the DBM and MBM comprised within thecomposition of the invention are of vertebrate origin, and they may beof human origin.

[0030] In a second embodiment, the DBM or MBM comprised within thecomposition of the invention are in powder or slice form. The particlesize of the DBM may be about 50 to 2500μ. Preferably, said particle sizeis about 250 to 500μ. The most preferable particle size will depend onthe specific needs of each case.

[0031] In another embodiment, the composition of the invention is forrestoring and/or enhancing the formation of a new hyaline cartilage andsubchondral bone structure.

[0032] In a further embodiment, the composition of the invention isintended for the treatment of a patient suffering from any one of ahereditary or acquired bone disorder, a hereditary or acquired cartilagedisorder, a malignant bone or cartilage disorder, conditions involvingbone or cartilage deformities and Paget's disease. Additionally, theinvention is also intended for the treatment of a patient in need of anyone of correction of complex fractures, bone replacement and formationof new bone in plastic or sexual surgery.

[0033] In a yet further embodiment, the composition of the invention mayfurther optionally comprise a pharmaceutically acceptable carrier ordiluent, as well as additional active agents.

[0034] In another aspect, the present invention relates to a method fortransplantation of a mixture comprising BMC with DBM and/or MBM andoptionally further comprising pharmaceutically acceptable carrier ordiluent, into a joint and/or a cranio-facial-maxillary bone of a subjectin need, wherein said method comprises introducing into said joint orbone the composition of the invention.

[0035] In a first embodiment of the method of the invention, the mixtureis administered by any one of the following procedures injection,minimally invasive arthroscopic procedure, or by surgical arthroplastyinto the site of implantation, wherein said method is for support orcorrection of congenital or acquired abnormalities of the joints,cranio-facial-maxillary bones, orthodontic procedures, bone or articularbone replacement following surgery, trauma or other congenital oracquired abnormalities, and for supporting other musculoskeletalimplants, particularly artificial and synthetic implants.

[0036] Thus, in a further aspect, the invention relates to a method oftreating a damaged or degenerative arthropathy associated withmalformation and/or dysfunction of cartilage and/or subchondral bone ina mammal in need of such treatment, comprising administering into anaffected joint or bone of said mammal a mixture comprising BMC with DBMand/or MBM, said mixture optionally further comprising apharmaceutically acceptable carrier or diluent and/or additional activeagents.

[0037] In one embodiment, the BMC which are present in the administeredmixture are either allogeneic or said mammal's own.

[0038] In another embodiment, the DBM or MBM which is present in theadministered mixture is in a slice, powder, gel, semi-solid or solidform embedded in or encapsulated in polymeric or biodegradablematerials.

[0039] In a yet further aspect, the present invention relates to anon-invasive (through injection), minimally invasive (througharthroscopy) or surgical transplantation method for support of implantsof joints or other musculoskeletal implants, comprising introducing agraft into a joint or a cranio-facial-maxillary bone of a subject inneed, wherein said graft comprises a mixture of BMC and DBM or MBM.

[0040] In an even further aspect, the present invention relates to theuse of a composition comprising BMC and DBM and/or MBM as a graft ofmesenchymal and/or mesenchymal progenitor cells fortransplantation/implantation into a mammal, wherein said mammal ispreferably a human. The transplantation is to be performed into a jointor into a cranio-facial-maxillary bone, for the development of new boneand/or cartilage.

[0041] Furthermore, the composition used in said transplantation isintended for the treatment of a patient suffering from any one of ahereditary or acquired bone disorder, a hereditary or acquired cartilagedisorder, a malignant bone or cartilage disorder, conditions involvingbone or cartilage deformities and Paget's disease. In addition, saidcomposition is intended for the treatment of a patient in need of anyone of correction of complex fractures, bone replacement and formationof new bone in plastic or sexual surgery.

[0042] In one embodiment, the composition used in the invention furthercomprises an active agent.

[0043] In another embodiment, the DBM and MBM comprised within thecomposition used in the invention are of vertebrate origin, and they maybe of human origin. Moreover, said DBM and MBM may be in powder, strips,thin layers, or slice form.

[0044] In an additional aspect, the present invention concerns the useof a mixture of BMC with DBM and/or MBM in the preparation of a graftfor the treatment of a bone or cartilage disorder.

[0045] Lastly, the present invention provides a kit for performingtransplantation into a joint or a cranio-facial-maxillary bone of amammal of BMC in admixture with DBM and/or MBM, wherein said kitcomprises:

[0046] (a) DBM and/or MBM in a compacted form;

[0047] (b) a BM aspiration needle;

[0048] (c) an intra-osseous bone drilling burr;

[0049] (d) a needle with a thick lumen for infusion of viscous bonemarrow-DBM mixture;

[0050] (e) a 2-way lumen connector for simultaneous mixing of BMC withDBM and diluent;

[0051] (f) a medium for maintaining BMC; and optionally

[0052] (g) cryogenic means for handling and maintaining BMC or BMCtogether with DBM.

[0053] The kit of the invention may optionally further comprise acarrier and/or a diluent for the BMC and DBM and/or MDM mixture.

BRIEF DESCRIPTION OF THE FIGURES

[0054] FIGS. 1A-L: Photomacrographs and micrographs of sagital kneejoint sections 2 to 24 weeks after the experimentally createdmicrofracture drilling defect (Picroindigocarmin, PIC, staining).

[0055]FIG. 1A: Photomacrograph of a normal rat knee joint section.

[0056]FIG. 1B: Photomicrograph of the entire normal osteo-chondralcomplex in the interchondylar region of the femur.

[0057]FIG. 1C: Photomicrograph of the articular cartilage in the normalosteo-chondral complex shown in FIG. 1B.

[0058]FIG. 1D: Microfracture drilling (full thickness defect),immediately after damage.

[0059]FIG. 1E: Micro-fracture left without the implant, two weeks afterdamage. The drilled hole, filled with connective tissue, can be seen.

[0060]FIG. 1F: Micro-fracture left without the implant, 24 weeks afterdamage. Regenerated subchondral bone and damaged joint surfaceconstituted of fibro-cartilaginous tissue can be seen.

[0061]FIG. 1G: DBM particles alone were transplanted into defect area,two weeks after transplantation. DBM particles are clearly seen in thesite of transplantation surrounded mostly with connective tissue.

[0062]FIG. 1H: DBM particles alone were transplanted into defect area,24 weeks after transplantation. Regenerated sub-chondral bone anddamaged joint surface covered with connective tissue together withfibro-cartilage could be observed.

[0063]FIG. 1I: DBM particles together with BMC were transplanted intodefect area, 2 weeks after transplantation. Extensively developinghyaline cartilage surrounding the implanted DBM particles could be seen.

[0064]FIG. 1J: DBM particles together with BMC were transplanted intodefect area, 4 weeks after transplantation. Extensively developinghyaline cartilage, as well as considerably degraded DBM particles can beseen.

[0065]FIG. 1K: DBM particles together with BMC were transplanted intodefect area, 8 weeks after transplantation. Almost complete regenerationof subchondral bone; surface of the damaged area is built of a continueslayer of extensively developing young hyaline cartilage.

[0066]FIG. 1L: DBM particles together with BMC were transplanted intodefect area, 24 weeks after transplantation. The histological structureof the regenerated osteo-chondral complex is indistinguishable fromnormal. Abbreviations: Typ. Kn. J., typical knee joint; Osteoch. Comp.,osteochondral complex; Norm. Cart., normal cartilage; Def., defect; D.,day(s); Al., alone; We., week(s); NROC, newly reconstitutedosteochondral complex.

[0067] FIGS. 2A-G: Laser Capture Microdissection and PCR analysis ofcells captured from the newly reconstituted osteochondral complex of theknee joint (6 months after transplantation of DBM with donor male BMCinto female recipient).

[0068]FIG. 2A: Laser shot general area, new cartilage formation.

[0069]FIG. 2B: Laser shot cap, new cartilage formation.

[0070]FIG. 2C: Magnification (×20) of laser shot cap, new cartilageformation.

[0071]FIG. 2D: Laser shot general area, new subchondral bone formation.

[0072]FIG. 2E: Laser shot cap, new subchondral bone formation.

[0073]FIG. 2F: Magnification (×20) of laser shot cap, new subchondralbone formation.

[0074]FIG. 2G: Detection of donor-derived cells by PCR analysis. Lanes:1, DNA size markers (φX714 cut with HaeIII), arrows point to the 194bp-long and 118 bp-long bands, respectively; 2, Amplification of malerat DNA derived from cartilage area of female rat knee joint; 3,Amplification of male rat DNA derived from subchondral bone area offemale rat knee joint; 4, Male rat DNA derived from hematopoietic marrowarea of female rat knee joint; 5, Internal positive control DNA frommale blood. The PCR results confirm the expression of donor derivedcells in all the three tissues composing the newly reconstitutedosteochondral complex. Abbreviations: Targ. Ar. LCM Kn. J., target areafor LCM in the knee joint; Las. Sh. Ar., laser shot area; Las. Sh. Ca.,laser shot cap; Ca. Marn., caps magnified; Cart., cartilage; S. Bo.,subchondral bone; Ost. Ch. Comp., osteo-chondral complex; Det. Don. Der.Cel. PCR Anal., detection of donor-derived cells by PCR analysis; Lan.,lanes.

[0075] FIGS. 3A-F: Correction of the calvarial defect by transplantationof demineralized bone matrix (DBM) and bone marrow cells (BMC) in rats,shown by sagital sections stained with Picroindigocarmin (PIC).

[0076]FIG. 3A: Photomacrographs of a normal rat cranium. Region markedby a square (D) is shown in FIG. 3B in higher magnification.

[0077]FIG. 3B: Site of the artificial defect (D) in the parietal regionof the cranium.

[0078]FIG. 3C: Photomacrograph of the defect area (DA) between the twocut edges.

[0079]FIG. 3D: Photomicrograph of cranial section 8 days after theexperimentally created calvarial defect (PIC staining). Defect leftuntreated. Cut edge (CE) and the fibrous connective tissue can be seen.

[0080]FIG. 3E: Photomicrograph of cranial section 8 days after theexperimentally created calvarial defect (PIC staining). DBM particlesalone were transplanted into defect area. Actively proliferatingfibroblastic cells surrounding the cut edge and DBM particles could beseen.

[0081]FIG. 3F: Photomicrograph of cranial section 8 days after theexperimentally created calvarial defect (PIC staining). DBM particlestogether with BMC were transplanted into defect area. Active remodelingof the transplanted DBM particles, areas of new bone formation areclearly visible. Abbreviations: Norm. Ra. Cran., normal rat cranium;Def., defect; Def. Ar., defect area; Def. Al., defect alone; D., day(s);Po. Transpl., post-transplantation.

[0082] FIGS. 4A-L: Photomacro- and micrographs of cranial sections 15and 30 days after the experimentally created calvarial defect (Sagitalsections, Picroindigocarmin, PIC, staining).

[0083]FIG. 4A: 15 days post-operation, control (no transplant).

[0084]FIG. 4B: 15 days post-transplantation, transplantation of DBMalone.

[0085]FIG. 4C: 15 days post-transplantation, transplantation of DBM andBMC.

[0086]FIG. 4D: 15 days post-operation, control (no transplant), 10×magnification. There is no new bone formation in the area of defect.

[0087]FIG. 4E: 15 days post-transplantation of DBM alone, 10×magnification. Remodeling of DBM particles results in bridging the areaof defect with the newly formed bone tissue.

[0088]FIG. 4F: 15 days post-transplantation of DBM and BMC, 10×magnification. The cut edge of the parietal bone could hardly bedistinguished in the continuous uniform layer of actively remodelingbony tissue.

[0089]FIG. 4G: 30 days post-operation, control (no transplant).

[0090]FIG. 4H: 30 days post-transplantation of DBM alone.

[0091]FIG. 4I: 30 days post-transplantation of DBM and BMC.

[0092]FIG. 4J: 30 days post-operation, control (no transplant), 10×magnification. There is no new bone formation in the area of defect.

[0093]FIG. 4K: 30 days post-transplantation of DBM alone, 10×magnification. Remodeling of DBM particles results in bridging the areaof defect with the newly formed bone tissue.

[0094]FIG. 4L: 30 days post-transplantation of DBM and BMC, 10×magnification. The cut edge of the parietal bone could hardly bedistinguished in the continuous uniform layer of actively remodelingbony tissue. Abbreviations: Def. Al., defect alone; Def., defect; D.,day(s); DA, area of defect; CE, cut edge.

[0095] FIGS. 5A-F: Laser Capture Microdissection (LCM) and PCR analysisof cells captured from the newly developing bony tissue in the area ofthe experimentally created calvarial defect after transplantation of DBMtogether with donor male BMC to female recipient.

[0096]FIG. 5A: General view of the normal rat cranium with the placewhere the defect was inflicted highlighted (D).

[0097]FIG. 5B: Regenerating bony tissue, area target for LCM.

[0098]FIG. 5C: Higher magnification, area target for LCM.

[0099]FIG. 5D: Laser shot caps; cells captured from this area were usedfor PCR analysis.

[0100]FIG. 5E: Laser shot caps, 10× magnification, cells captured forPCR analysis.

[0101]FIG. 5F: Detection of donor-derived cells by PCR analysis. Lanes:1, DNA size markers (φX714 cut with HaeIII), arrows point to the 194bp-long and 118 bp-long bands, respectively; 2, male rat DNA derivedfrom bone area of female knee cranium; 3, internal positive control, DNAfrom male blood. The results of the PCR analysis confirm the expressionof donor derived cells in the newly forming bony tissue. Abbreviations:Norm. Ra. Cran., normal rat cranium; Bo., bone; Las. Sh. Ar., laser shotarea; Las. Sh. Ca., laser shot caps; Ca. Magn., caps magnified; Targ.Ar. LCM, target area for LCM; CE, cut edge; D, area of defect; Det. Don.Der. Cel. PCR Anal., detection of donor-derived cells by PCR analysis;bp, base-pair(s).

DETAILED DESCRIPTION OF THE INVENTION

[0102] The following abbreviations are utilized throughout thisspecification:

[0103] BM: bone marrow

[0104] BMC: bone marrow cell(s)

[0105] BMP: bone morphogenetic protein

[0106] DBM: demineralized bone matrix

[0107] LCM: Laser Capture Microdissection

[0108] MBM: mineralized bone matrix

[0109] PCR: polymerase chain reaction

[0110] PIC: Picroindigocarmin, a dye used in histological staining.

[0111] In search for improving regeneration of damaged osteochondralcomplex in joint and cranio-facial-maxillary areas, the inventors havefound that using a composition comprising BMC and DBM and/or MBM as agraft results in the development of bone and cartilage according to thelocal conditions of the site of transplantation. New tissue formationfollows a differentiation pathway producing different types of bone andcartilage, depending on the local conditions. Thus, the newly formedtissue meets precisely the local demands.

[0112] The present invention relates to compositions comprising amixture of bone marrow cells (BMC) and demineralized and/or mineralizedbone matrix (DBM and MBM, respectively) and to their novel uses in thetransplantation of mesenchymal progenitor cells into joints andcranio-facial-maxillary bones.

[0113] Thus, in a first aspect, the present invention relates to acomposition comprising bone marrow cells (BMC) and demineralized bonematrix (DBM) and/or mineralized bone matrix (MBM).

[0114] DBM is a preferable essential ingredient in the composition ofthe invention in view of its advantageous ability to combine all thefeatures needed for making it an excellent carrier for mesenchymalprogenitor cells. The properties of DBM can be summarized as follows:

[0115] 1. DBM can be a conductive scaffold essential for theengraftment, proliferation and differentiation of mesenchymal progenitorcells, in the course of bone and cartilage formation.

[0116] 2. DBM is the natural source of BMPs, which are active instimulating osteo- and chondrogenesis, thus also fulfilling theinductive function.

[0117] 3. DBM is slowly biodegradable, the degradation time beingcompatible with the period of de novo chondro- and osteogenesis.

[0118] 4. DBM has very low immunogenicity when used as a xenograft, andit is practically non-immunogenic when used in allogeneic combinations.

[0119] 5. DBM is sufficiently flexible and strong to providebiomechanical properties to the joint surface during the period of newcartilage formation.

[0120] 6. DBM can be provided as an amorphous powder that can beinjected locally, without major surgical intervention, thus avoidingiatrogenic damage to complex joints.

[0121] In a second aspect, said composition comprising BMC and DBMand/or MBM is for use in transplantation of mesenchymal cells and/ormesenchymal progenitor cells into a joint and/or acranio-facial-maxillary area of a subject in need, wherein said subjectis a mammal, preferably a human.

[0122] It is an object of the present invention to provide the saidcomposition for transplantation of BMC into damaged joints for thereplacement and/or restoration of hyaline cartilage as well as ofsubchondral bone, originating from the mesenchymal precursor cellsexisting in the transplanted BMC.

[0123] In a first embodiment, the DBM and MBM comprised within thecomposition of the invention are of vertebrate origin, and they may beof human origin.

[0124] In a second embodiment, the DBM and MBM comprised within thecomposition of the invention are in powder or slice form. The particlesize of the DBM may be about 50 to 2500μ. Preferably, said particle sizeis about 250 to 500μ. The most preferable particle size will depend onthe specific needs of each case.

[0125] In another embodiment, the composition of the invention is forrestoring and/or enhancing the formation of a new hyaline cartilage andsubchondral bone structure.

[0126] The idea underlying the present invention is that bone marrowcells (BMC) may provide a source for mesenchymal stem cells, which arecapable of inducing osteo- and chondrogenesis. Thus, as described in thefollowing examples, when a BMC suspension in admixture with DBM and/orMBM powder was administered directly into either a joint bearing adamage in the osteo-chondral complex, or in the cranium of an animalwith a partial bone defect in the parietal bone, significant restorationoccurred. Treated recipients were mobile with no need for fixation ofthe joints, and full restoration of the anatomic structure of thetreated joint was accomplished. Likewise, newly reconstituted parietalbone replacing surgically removed parietal bone in the skull showednormal remodeling. In the damaged joint, there was formation ofsubchondral bone structure and hyaline cartilage, and in the cranialdefect, new flat bone was formed, both originating from the mesenchymalcells present in the transplanted BMC, as confirmed by the LCM-PCRanalysis.

[0127] In a further embodiment, the composition of the invention isintended for the treatment of a patient suffering from any one of ahereditary or acquired bone disorder, a hereditary or acquired cartilagedisorder, a malignant bone or cartilage disorder, metabolic bonediseases, bone infections, conditions involving bone or cartilagedeformities and Paget's disease. Said disorders are listed in detail inTable 1. Additionally, the invention is also intended for the treatmentof a patient in need of any one of correction of complex fractures, bonereplacement, treatment of damaged or degenerative arthropathy andformation of new bone in plastic or sexual surgery. TABLE 1 Congenitaland Non-neoplastic Hereditary Metabolic Disorders Bone Disorders BoneInfections Bone Diseases of the Bone Achondroplasia HematogenousOsteoporosis Fibrous Dysplasia of (Pyogenic) the Bone OsteomyelitisOsteogenesis Osteomyelitis Rickets and Fibrous Cortical Imperfecta froma Osteomalacia Defect and Non- (Brittle Bones, Contiguous ossifyingFibroma Fragilitas Infection Ossium) Osteopetrosis Osteomyelitis BoneChanges Solitary Bone Cyst (Marble Bone from an in (Unicameral BoneDisease, Introduced Hyperparathy Cyst) Osteosclerosis) Infection roidism(Generalized Osteitis, Cystic Fibrosis, Von Recklinghausen's BoneDisease) Hereditary Bone Renal Aneurysmal Bone Multiple TuberculosisOsteodystrophy Cyst Exotosis (Osteochondro- matosis) Enchondro- BoneSyphilis Paget's Eosinophilic matosis Disease of Granuloma of Bone(Oilier's Bone (Osteitis Disease) Deformans) Bone Fungus Bone Lesions ofInfections Gaucher's Disease

[0128] In a yet further embodiment, the composition of the invention mayfurther optionally comprise a pharmaceutically acceptable carrier ordiluent, as well as additional active agents.

[0129] A pharmaceutically acceptable (or physiologically acceptable)additive, carrier and/or diluent mean any additive, carrier or diluentthat is non-therapeutic and non-toxic to recipients at the dosages andconcentrations employed, and that does not affect the pharmacological orphysiological activity of the active agent.

[0130] The preparation of pharmaceutical compositions is well known inthe art and has been described in many articles and textbooks, see e.g.,Remington's Pharmaceutical Sciences, Gennaro A. R. ed., Mack PublishingCompany, Easton, Pa., 1990, and especially pages 1521-1712 therein.

[0131] Active agents of particular interest are those agents thatpromote tissue growth or infiltration, such as growth factors. Oneexample is BMPs, which may enhance the activity of the composition ofthe invention. Other exemplary growth factors for this purpose includeepidermal growth factor (EGF), osteogenic growth peptide (OGP),fibroblast growth factor (FGF), platelet-derived growth factor (PDGF),transforming growth factors (TGFs), parathyroid hormone (PTH), leukemiainhibitory factor (LIF), insulin-like growth factors (IGFs), and growthhormone. Other agents that can promote bone growth, such as theabove-mentioned BMPs, osteogenin [Sampath et al. (1987) Proc. Natl.Acad. Sci. USA 84:7109-13] and NaF [Tencer et al. (1989) J. Biomed. Mat.Res. 23: 571-89] are also preferred.

[0132] Other active agents may be anti-rejection or tolerance inducingagents, as for example immunosupressive or immunomodulatory drugs, whichcan be important for the success of bone marrow allografts or xenograftstransplantion.

[0133] Alternatively, said active agents may be for example antibiotics,provided to treat and/or prevent infections at the site of the graft. Onthe same token, anti-inflammatory drugs can also be added to thecomposition of the invention, to treat and/or prevent inflammations atthe site of the graft. Said inflammations could be the result of forexample rheumatoid arthritis, or other conditions.

[0134] Polymeric or biodegradable materials are pharmaceuticallyacceptable carriers and diluents. Biodegradable films or matrices,semi-solid gels or scaffolds include calcium sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid, polyanhydrides, bone ordermal collagen, fibrin clots and other biologic glues, pure proteins,extracellular matrix components and combinations thereof. Suchbiodegradable materials may be used in combination withnon-biodegradable materials, to provide desired mechanical, cosmetic ortissue or matrix interface properties.

[0135] In preferred embodiments, the composition of the inventioncontains BMC suspensions at cell concentrations ranging from 1×10⁶/ml to1×10⁸/ml and DBM at a ratio of from 1:1 to 20:1, preferably between 2:1to 9:1, most preferably the composition of the invention is at a ratioof 4 parts BMC concentrate to 1 part of DBM in powder form(volume:volume). The absolute number of BMC and DBM is dependent on thesize of the joint that needs to be corrected or the size (surface, shapeand thickness) of the bone that needs to be replaced

[0136] In another aspect, the present invention relates to a method fortransplantation of a mixture comprising BMC with DBM and/or MBM andoptionally further comprising pharmaceutically acceptable carrier ordiluent, into a joint and/or a cranio-facial-maxillary bone of a subjectin need, wherein said method comprises introducing into said joint orbone the composition of the invention.

[0137] The composition of the invention, which possesses all theessential features for accomplishing local bone formation wherever it isimplanted, could be efficiently applied for all kinds of bone repair orsubstitution, especially in places lacking or deprived of mesenchymalstem cells. Amongst the most problematic places in this sense arejoints, cranio-facial-maxillary areas and different kinds of segmentalbony defects. Thus, the present invention may be explained as a complexgraft, comprising all necessary components, and which its implantationinto a damaged joint or bone is sufficient for regeneration orsubstitution of removed, damaged or destroyed cartilage and/or bone.

[0138] In a first embodiment of the method of the invention, the mixtureis administered by any one of the following procedures, injection,minimally invasive arthroscopic procedure, or by surgical arthroplastyinto the site of implantation, wherein said method is for support orcorrection of congenital or acquired abnormalities of the joints,cranio-facial-maxillary bones, orthodontic procedures, bone or articularbone replacement following surgery, trauma or other congenital oracquired abnormalities, and for supporting other musculoskeletalimplants, particularly artificial and synthetic implants.

[0139] Thus, in a further aspect, the invention relates to a method oftreating a damaged or degenerative arthropathy associated withmalformation and/or dysfunction of cartilage and/or subchondral bone ina mammal in need of such treatment, comprising administering into anaffected joint or bone of said mammal a mixture comprising BMC with DBMand/or MBM, said mixture optionally further comprising apharmaceutically acceptable carrier or diluent and/or additional activeagents.

[0140] As demonstrated in the following examples, the process of induceddevelopment (i.e. proliferation and differentiation) of mesenchymalprogenitor cells present within the BMC/DBM mixture can accomplish boneand cartilage formation wherever the mixture is transferred to. Thefindings presented by the inventors indicate that administration of thecomposition of the invention into a damaged area of the joint, resultsin generation of new osteochondral complex consisting of articularcartilage and subchondral bone. When administered into an experimentallycreated calvarial defect, the composition of the invention results ingeneration of full intramembranous bone development at the site oftransplantation. New tissue formation follows a differentiation pathway,producing different types of bone and cartilage, depending on the localconditions. Thus, the newly formed tissue meets precisely the localdemands.

[0141] In one embodiment, the BMC which is present in the administeredmixture are either allogeneic or said mammal's own.

[0142] In another embodiment, the DBM or MBM which is present in theadministered mixture is in a slice, powder, gel, semi-solid or solidform embedded in or encapsulated in polymeric or biodegradablematerials.

[0143] The procedure of applying the composition of the invention into adamaged joint or cranial area comprises the following steps:

[0144] 1. Selecting the source for BMC. The donor may be allogeneic orthe BMC may be obtained from the same treated subject (autologoustransplantation).

[0145] 2. Selecting the source of DMB and/or MBM. The DBM may besupplied commercially and since it is not immunogenic, there are nolimitations for a specific donor. DMB and/or MBM may be in powder,granules or in slice form. The particle size of the DBM may be about 50to 2500μ. Preferably, said particle size is about 250 to 500μ. The mostpreferable particle size will depend on the specific needs of each case.

[0146] 3. Preparing a composition comprising a suspension of BMC, at acell concentration ranging from 1×10⁶/ml to 1×10⁸/ml and mixing it withDBM at a ratio of from 1:1 to 20:1, preferably between 2:1 to 9:1, mostpreferably the composition of the invention is at a ratio of 4 parts BMCconcentrate to 1 part of DBM in powder form (volume:volume). MDM may beused instead of DBM. If so desired, BMP may optionally be included inthe composition.

[0147] 4. Administering said composition into a subject in need eitherthrough a syringe (non-invasive injection), closed arthroscopy or opensurgical procedure. Alternatively, the composition may be administeredso that it is encapsulated within normal tissue membranes. Stillalternatively, the composition may be contained within a membranousdevice, made of a selective biocompatible membrane that allows cells,nutrients, cytokines and the like to penetrate the device, and at thesame time retains the DBM and/or MBM particles within the device. Such amembranous device, bone strips or additional scaffolds are preferablysurgically introduced. Or, still alternatively, the composition may beadministered within a biocompatible and biodegradable polymeric deviceretaining the DBM and/or MBM particles within the device and suitable tocreate the needed shape of the transplanted complex.

[0148] 5. Providing glue, preferably consisting of fibrinogen andthrombin, this may be used for fixation of the implant composition atthe site of implantation, if necessary.

[0149] In a yet further aspect, the present invention relates to anon-invasive transplantation method comprising introducing a graft intoa joint or a cranio-facial-maxillary bone of a subject in need, whereinsaid graft comprises a mixture of BMC and DBM or MBM.

[0150] In the examples presented herein (see Examples), the inventorsshow that administration of the composition of the present invention(e.g. BMC in admixture with DBM, as in Example 1) into a damaged area ofthe joint is essential and sufficient for the generation of newosteochondral complex, consisting of articular cartilage and subchondralbone, at the site of transplantation. The newly formed donor-derivedosteochondral complex was capable of long-term maintenance, remodelingand self-renewal, as well as carrying out specific functions of jointsurface, such as motion and weight bearing.

[0151] In an even further aspect, the present invention relates to theuse of a composition comprising BMC and DBM and/or MBM as a graft ofmesenchymal and/or mesenchymal progenitor cells for transplantation intoa mammal, wherein said mammal is preferably a human. The transplantationis to be performed into a joint or into a cranio-facial-maxillary bone,for the development of new bone and/or cartilage. The graft of saidtransplantation may also be for supporting orthodontical procedures forbone augmentation caused by aging, or by congenital, acquired ordegenerative processes.

[0152] Furthermore, the composition used in said transplantation isintended for the treatment of a patient suffering from any one of ahereditary or acquired bone disorder, a hereditary or acquired cartilagedisorder, a malignant bone or cartilage disorder, conditions involvingbone or cartilage deformities and Paget's disease. In addition, saidcomposition is intended for the treatment of a patient in need of anyone of correction of complex fractures, bone replacement, treatment ofdamaged or degenerative arthropathy and formation of new bone in plasticor sexual surgery.

[0153] The method of the invention may also be used to induce or improvethe efficiency of bone regeneration in damaged cranio-facial-maxillaryareas, for therapeutic and cosmetic purposes.

[0154] In one embodiment, the composition used in the invention furthercomprises an additional active agent.

[0155] In another embodiment, the DBM and MBM comprised within thecomposition used in the invention are of vertebrate origin, and they maybe of human origin. Moreover, said DBM and MBM is in powder or sliceform.

[0156] In an additional aspect, the present invention concerns the useof a mixture of BMC with DBM and/or MBM in the preparation of a graftfor the treatment of a bone or cartilage disorder, and/or for support ofmusculoskeletal implants, as a ‘glue’ to enforce metal implants, joints,etc. that may become lose with time, or to provide a constantly adapting“biological glue” to support such non-biological implants.Alternatively, the invention could be for the support of limbtransplants, especially in the articular/bone junction.

[0157] Lastly, the present invention provides a kit for performingtransplantation into a joint or a cranio-facial-maxillary bone of amammal of BMC in admixture with DBM and/or MBM, wherein said kitcomprises:

[0158] (a) DBM and/or MBM in a compacted form;

[0159] (b) a BM aspiration needle;

[0160] (c) an intra-osseous bone drilling burr;

[0161] (d) a needle with a thick lumen for infusion of viscous bonemarrow-DBM mixture;

[0162] (e) a 2-way lumen connector for simultaneous mixing of BMC-DBMand diluent;

[0163] (f) a medium for maintaining BMC; and optionally

[0164] (g) cryogenic means for handling and maintaining BMC or BMCtogether with DBM.

[0165] The kit of the invention may optionally further comprise acarrier and/or a diluent for the BMC and DBM and/or MDM mixture.

[0166] The present inventors have concluded that transplantation ofmultipotent mesenchymal stem cells, and not of differentiated bone orchondrocytes, for remodeling and restoration of a healthy joint orcranio-facial-maxillary structure in arthropathy, is especiallyimportant for the following reasons:

[0167] (1) Chondrocytes, as well as the cells transferred within a bonetransplant are already fully differentiated cells, with relatively lowmetabolic activity and limited self-renewal capacity that may besufficient to maintain healthy cartilage or bone, but is certainlyinsufficient for the development of large areas of bone or of hyalinecartilage de novo.

[0168] (2) Most frequently in joints, both cartilage and subchondralbone are damaged. Thus, even a successfully developed new hyalinecartilage is unlikely to be maintained for long if the subchondral boneis left damaged. Based on these findings, it was observed in thefollowing examples that mesenchymal stem cells present in bone marrow,if transplanted under the appropriate conditions, will create aself-supporting osteochondral complex providing healthy joint surface.

[0169] It is not yet clear what makes multipotential mesenchymal stemcells, under the influence of DBM, to choose between an osteogenic and achondrogenic differentiation pathway. It has however been reported thatthe ratio of cartilage to bone production depends in particular on thesite of DBM implantation, which is naturally influenced by the localconditions [Inoue, T. et al. (1986) J Dent Res 65(1):12-22], such as thelocal source of mesenchymal cells and blood supply [Reddi, A. H. andHuggins, C. H. (1973) P.S.E.B.M. 143:634-637]. Low oxygen tension favorschondrogenesis [Bassett, C. A. L. (1962) J Bone Joint Surg 44A:1217],most likely due to the low O₂ tension in poorly vascularized cartilage[Sledge, C. B. and Dingle, J. T. (1965) Nature (London) 205: 140].Interestingly, a successful substitution of anterior cruciate ligament(ACL) by demineralized cortical bone matrix has been reported in a goatmodel [Jackson, D. W. et al. (1996) Amer J Sports Medicine24(4):405-414]. The remodeling process included new bone formationwithin the matrix in the osseous tunnels and a ligament-like transitionzone developing at the extra-articular tunnel interface [Jackson, D. W.et al. (1996) id ibid.]. Taking into consideration that hyalinecartilage is naturally developed and maintained only in the joints,where contact with synovial membranes and lubrication with synovialfluid is available and probably essential, it seems reasonable to assumethat the environmental conditions in the joint play a major role inenhancing chondrogenesis.

[0170] In the following examples the inventors have shown, for the firsttime, that a graft composed of DBM and/or MBM and bone marrow cellstransplanted into a damaged joint or cranial bone, led to successfulreplacement of damaged cartilage and subchondral bone. This was theresult of osteogenesis on the side of contact with bone andchondrogenesis on the free joint surface, thus the physiologicalenvironmental conditions favored osteogenesis or chondrogenesis,respectively. The same kind of a graft composed of DBM and/or MBM andbone marrow cells transplanted into experimentally created partial bonedefect in the parietal bone of the cranium led to successful replacementof the removed part of the bone. Thus, the new tissue formation followsa differentiation pathway, producing different types of bone andcartilage depending on the local conditions, such that the newly formedtissue meets precisely the local demands.

[0171] Many publications are referred to throughout this application.The contents of all of these references are fully incorporated herein byreference.

[0172] Throughout this specification and the claims which follow, unlessthe context requires otherwise, the word “comprise”, and variations suchas “comprises” and “comprising”, will be understood to imply theinclusion of a stated integer or step or group of integers or steps butnot the exclusion of any other integer or step or group of integers orsteps.

[0173] It must be noted that, as used in this specification and theappended claims, the singular forms “a”, “an” and “the” include pluralreferents unless the content clearly dictates otherwise.

[0174] The following examples are representative of techniques employedby the inventors in carrying out aspects of the present invention. Itshould be appreciated that while these techniques are exemplary ofpreferred embodiments for the practice of the invention, those of skillin the art, in light of the present disclosure, will recognize thatnumerous modifications can be made without departing from the spirit andintended scope of the invention.

EXAMPLES Experimental Procedures

[0175] 1. Animals

[0176] 8 weeks old C57BL/6 male mice and Lewis male rats with bodyweight of 180-200 g were used as the donors of bones (for matrixpreparation) and BMC. Rats from the same batches were used as graftrecipients.

[0177] 2. Preparation of Demineralized Bone Matrix (DBM)

[0178] Demineralized bone matrix (DBM) was prepared as described [Reddiand Huggins (1973) id ibid.] with the inventors' modification.Diaphyseal cortical bone cylinders from Lewis rats were cleaned frombone marrow and surrounding soft tissues, crumbled and placed in a jarwith magnetic stirring. Bone chips were rinsed in distilled water for2-3 hrs; placed in absolute ethanol for 1 hr and in diethyl ether for0.5 hr, then dried in a laminar flow, pulverized in a mortar with liquidnitrogen and sieved to select particles between 400 and 1,000μ. Theobtained powder was demineralized in 0.6M HCl overnight, washed forseveral times to remove the acid, dehydrated in absolute ethanol anddiethyl ether and dried.

[0179] Mineralized bone matrix (MBM) was prepared according to the sameprocedure but the stage of demineralization with HCl was omitted.

[0180] With the exception of the drying step, all steps of the procedurewere performed at 4° C.; to prevent degradation of Bone MorphogeneticProteins (BMP) by endogenous proteolytic enzymes. The matrices werestored at −20° C.

[0181] 3. Preparation of the Implanted Material

[0182] Preparation of Donor BMC Suspensions for Transplantation:

[0183] The femurs of donor mice or rats were freed of muscle. Marrowplugs were mechanically pressed out of the femoral canal by a mandrin.Highly concentrated single cell suspensions of BMC were prepared bydissolving 4-5 femoral plugs into 100 μl of RPMI 1640 medium (BiologicalIndustries, Beit Haemek, Israel), and passing the cells through theneedle several times to dissolve the bone marrow tissue into asingle-cell suspension. The number of nucleated cells per femoral bonemarrow plug is rather stable (about 10⁷ cells/plug for a C57BL/6 male, 8week old mouse). Several reproducible verifications have shown that BMCprepared for transplantation in a form of a single cell suspensioncontains an approximate concentration of 3×10⁸ cells/ml.

[0184] Composition of the Grafts:

[0185] Grafts were composed of the following ingredients, in differentcombinations:

[0186] 1. 20 μl of BMC suspension (concentration 3×10⁸ cells/ml);

[0187] 2. 4 mg of DBM (or MBM);

[0188] 3. 0.5 μg BMP-2 (R & D systems, USA), optionally.

[0189] The exogenous BMP that is optionally added to the composition ofthe invention is not a mandatory ingredient. DBM exhibits conductiveproperties essential for the engraftment, proliferation anddifferentiation of mesenchymal progenitor cells transplanted within BMCsuspension, in the course of bone and cartilage formation. At the sametime, DBM is the natural source of BMPs (bone morphogenetic proteins)active in stimulating osteo- and chondrogenesis, thus fulfilling alsothe inductive function. Addition of exogenous BMPs may enhance theefficiency of the induction.

[0190] 4. Implantation of a Mixture of BMC and Demineralized (orMineralized) Bone Matrix Into the Area of Local Damage in the ArticularCartilage of the Knee Joint

[0191] A standard artificial damage in the articular cartilage andsubchondral bone in the rat knee joint was induced as described.Following anesthesia, the knee joint was accessed by a medialparapatellar incision, and the patella was temporarily displaced towardsthe side. A microfracture drilling (for a full thickness defect) of 1.5mm in diameter and 2.0 mm in depth was made in the interchondylar regionof the femur.

[0192] The defect was filled with DBM (or MBM) in the form of powder(with particle size of 300-450 micron, or in the form pf slice), alone(control) or together with the BMC suspension, prepared as describedabove. Another control consisted of transferring only BMC into thedamaged area. The transplanted material was fixed in place withfibrinogen-thrombin tissue adhesive glue, the patella was returned intoits place and the incision was sutured with bioresorbable thread. Theskin was closed with stainless clips. In another control group, thedamaged area was closed with fibrinogen-thrombin tissue adhesive glueonly, without the addition of any of DBM, MBM or BMC.

[0193] 5. Implantation of a Mixture of BMC and Demineralized Bone MatrixInto the Experimentally Created Calvarial Defect.

[0194] Male Lewis rats were anesthetized by intraperitoneal injection ofKetamine. An incision was performed in the frontal region of the ratcranium. The muscular flap was removed from the parietal bone area and abony defect (0.4×0.5 mm²) was made lateral to the saggital suture usinga dental burr. The defect was then filled with DBM in powder form(particle size of 300-450 micron) together with BMC suspension, preparedas described above. In one control group, only DBM particles weretransferred into the damaged area. The transplanted material was fixedin place with fibrinogen-thrombin tissue adhesive glue. In anothercontrol group the damaged area was only closed with fibrinogen-thrombintissue adhesive glue, without addition of the transplanted material. Theskin was closed with stainless clips.

[0195] 6. Laser Capture Microdissection (LCM) and Polymerase ChainReaction (PCR) Analysis of the Reconstituted Bone Articular Cartilageand Hematopoietic Tissue After Implantation of BMC and DemineralizedBone Matrix Into Damaged Intracondylar Region of Femoral Bone or Intothe Cranial Defect

[0196] In the experiments in which BMC of male donors was transplantedeither into the damaged intracondylar region of femoral bone, or intothe cranial defect, both in female recipients, the newly formedarticular cartilage, cranial and subchondral bone were checked by PCRanalysis for the origin of the donor. The new technology ofLaser-Capture Microdissection (LCM) (service provided by the CommonFacility Unit of Hadassah University Hospital, Jerusalem, Israel) allowsthe isolation of individual cells from tissue sections under precisemicroscopic control, and was used to harvest isolated cells from newlyformed articular cartilage, cranial and subchondral bone. PCR analysisof the harvested cells (about 70-100 cells per test) was performed usinga set of primers specific to the Sry gene—the sex determination regionof the Y chromosome [An, J. et al. (1997) J Androl. 18(3):289-93].

[0197] 7. Histological Evaluation

[0198] The autopsied material was fixed in 4% neutral bufferedformaldehyde, decalcified, passed through a series of ethanol grades andxylene, and then embedded in paraffin. Serial sections (5-7 micronsthick) were obtained. One set of representative serial sections of eachsample was stained with Hematoxylin & Eosin (H&E), and another one withPicroindigocarmin (PIC).

Example 1

[0199] Transplantation of BMC Into the Joint Together with Demineralized(or Mineralized) Bone Matrix

[0200]FIG. 1 presents the results of experiments carried out to testwhether the mesenchymal stem cells present within the bone marrow cellsof the composition of the invention could be induced to develop hyaline(articular) cartilage and subchondral bone, when transplanted into thedamaged areas of the knee joints.

[0201] Male Lewis rats were anesthetized by intraperitoneal injection ofKetamine. Microfracture drilling (full thickness defect) was inflictedin articular cartilage and subchondral bone in the interchondylar regionof the femur. The defects were then filled with DBM (or MBM) togetherwith BMC. In separate groups of experimental animals with defects inarticular cartilage and subchondral bone, said defects were filled withDBM (or MBM) or BMC alone. The optional addition of BMPs was also tested(data not shown). The implanted material was fixed in place withfibrinogen-thrombin tissue adhesive glue. In one group of controlanimals, only glue (and no DBM, MBM or BMC) was grafted into the defectarea.

[0202]FIGS. 1A, 1B and 1C show healthy undamaged knee joint of the ratwith osteo-chondral complex in the interchondylar region of the femur.In FIG. 1D, the microfracture drilling (full thickness defect) can beseen immediately after damage. When DBM powder mixed with BMC wastransplanted into the drilled hole, areas of extensively developinghyaline cartilage surrounding implanted DBM particles were observedalready two weeks after transplantation, when slight degradation of DBMparticles could be observed (FIG. 1I). One month after transplantationthe DBM particles were already considerably degraded, and areas ofextensively developing hyaline cartilage surrounding implanted DBMparticles were still present (FIG. 1J). After two months, regenerationof the subchondral bone was almost complete, while the surface of thedamaged area was built of a thick and continuous layer of extensivelydeveloping young hyaline (articular) cartilage (FIG. 1K). Six monthsafter transplantation of DBM powder, mixed with BMC, into themicrofracture drilling defect in the interchondylar region of the femur,the histological structure of the regenerated osteo-chondral complex wasindistinguishable from normal (FIG. 1L).

[0203] PCR analysis of isolated cells from different tissues composingnewly developed osteo-chondral complex, captured by LCM techniques afterimplantation of DBM together with BMC into the micro-fracture drillingwas performed (FIGS. 2A-F). The results of the PCR showed the presenceof donor derived cells within newly formed articular cartilage andsubchondral bone (FIG. 2G). This is strong evidence that activemesenchymal progenitor cells, transplanted within the donor BMCsuspension, took an active part in the development of a newosteo-chondral complex.

[0204] Most importantly, bone and cartilage regeneration, to the sameextent as that of the experimental group that received a DBM togetherwith BMC graft, were not observed in the control groups. Thus, theinventors concluded that the DBM+BMC mixture included the entire arrayof components essential for the successful regeneration of theosteo-chondral complex in the damaged joint.

[0205] The specificity of the artificial defect model used in thepresent experiments resided in the penetration of the microfracturedrill into the subchondral bone, thus supplying the damaged area withlocally existing bone marrow containing mesenchymal progenitor cellspotentially capable of restoring both subchondral bone and articularcartilage, when local conditions stimulating osteo- and chondrogenesiswere supplied.

[0206] However, without the implant, regeneration of micro-fracture wasincomplete, and two weeks after drilling the hole was filled withconnective tissue (FIG. 1E). During the following month, the subchondralbone was repaired, although with no regeneration of the articularcartilage on the surface of the damaged area (data not shown). Sixmonths after the micro-fracture had been inflicted, the regeneratingsurface of the damaged joint constituted of fibro-cartilaginous tissue(FIG. 1F). Implantation of exogenous BMC does not bring any considerablechanges in the pathway of regeneration, i.e. the regenerating surface ofthe damaged joint constituted of fibro-cartilaginous tissue usuallydeteriorating over time. It is important to note that, unfortunately,despite the restricted efficiency of this procedure, as also shown bythese results, this is the most used procedure for damaged joint surfacerepair and for the treatment of osteoarthritis in the current clinicalpractice.

[0207] When DBM particles alone were transplanted into the drilled hole,it seems that the number of locally available mesenchymal progenitorcells was not sufficient for effective regeneration of osteo-chondralcomplex. No extensive developing hyaline cartilage could be seen amongthe implanted DBM particles two weeks after transplantation (FIG. 1G).In most of the cases the degradation and remodeling of DBM particles aswell as the process of new bone- and cartilage formation was notefficient enough and the damaged area was finally covered withconnective tissue together with fibro-cartilage (FIG. 1H).

Example 2

[0208] Transplantation of BMC Together with DBM Into the ExperimentallyCreated Calvarial Defect.

[0209] Experiments were carried out to test whether the mesenchymal stemcells within the BMC comprised in the composition of the invention couldinitiate and accomplish the intramembranous development of bone, whentransplanted together with DBM into the experimentally created calvarialdefect. The results of these experiments are shown in FIGS. 3 and 4.This method could then be extended to treat facial-maxillary defects.

[0210] An incision was performed in the frontal cranium region ofanesthetized Lewis rats (8-12 weeks old) and the skin flap was movedaside. The muscular flap was removed from the parietal bone area and abony defect (0.4×0.5 mm²) was created laterally to the sagital sutureusing a dental burr. The defect area was either left empty, filled withDBM alone, or filled with DBM together with BMC, as described above. Inall the groups (experimental and control) the defect area was finallycovered with fibrin glue. Lastly, the skin flap was returned to placeand fixed with stainless clips.

[0211] The utilization of non-healing cranial defects allows for theobservation of both osteo-conductive and osteo-inductive components ofthe healing process. Thus, the non-healing cranial defect represents anappropriate model for evaluating the ability of the composition of thepresent invention (in this Example, BMC together with DBM) to accomplishintramembranous bone formation when transplanted into a damaged area ofthe crania.

[0212]FIG. 3A shows the normal (undamaged) cranium of a rat. FIGS. 3Band 3C show the experimental defect in the parietal bone area.

[0213] 15 and 30 days after the operation, absence of bony tissueregeneration could be observed when the site of removed bone was leftempty (FIG. 4A, 4D, 4G and 4J), suggesting that the size of the defectsufficiently large, compatible with the definition of non-healingcranial defect.

[0214] Filling of the experimental cranial defect with DBM aloneresulted in the gradual degeneration and remodeling of transplanted DBMparticles, and blood vessels and new bone formation on different stagesof maturity could be observed (FIG. 3E, FIG. 4B, 4E, 4H and 4K).However, the process of intensive new bone formation was not presenteduniformly in the defect area. New bone formation was considerably moreactive in the periphery, close to the edges of the cut bone, suggestingthat the number of mesenchymal progenitor cells that could be inducedand conducted to osteogenesis was limited in the central area of thedefect.

[0215] When DBM powder together with BMC was transplanted into the siteof the experimental cranial defect, extensive remodeling of thetransplanted DBM particles and developing areas of new bone could beobserved as early as 8 days after transplantation (FIG. 3F). 15 andmostly 30 days after transplantation, the cut edge of the parietal bonecould hardly be distinguished from the surrounding new bony tissue(FIGS. 4F and 4L). The defect area was reconstituted with a continuouslayer of newly developing bone (FIG. 4C, 4F, 4I and 4L). It should beespecially stressed that extensive remodeling of transplanted DBMparticles and active new bone formation were presented uniformlythroughout the defect area, suggesting that the quantity of availablemesenchymal progenitor cells capable of being induced and conducted toosteogenesis was sufficient when the implant consisted of DBM particlesmixed with BMC.

[0216] PCR analysis of the cells isolated by LCM from the newlydeveloped bone tissue, in the site of experimental calvarial damage, 8and 30 days after transplantation of DBM together with BMC (from maledonor to female recipient) showed the presence of donor derived cells(FIG. 5). This is strong evidence that the mesenchymal progenitor cellstransplanted within the donor BMC suspension play an active role in thedevelopment of this new cranial bone, and are subject to theosteo-inductive and osteo-conductive properties of DBM.

[0217] These findings indicate that administration of the composition ofthe present invention (in this case, DBM together with BMC) into anexperimentally created calvarial defect was sufficient for active andcomplete intramembranous bone formation at the site of transplantation.This procedure could be extended to treat facial-maxillary defects.

[0218] Pilot experiments utilizing BMC in combination with MBM ratherthan DBM also showed positive results. Mainly, the difference betweenemploying DBM and MBM lies on delayed bone and cartilage formation withMBM. Also, since MBM particles are much more dense and hard, as comparedto DBM particles, they are more useful when weight bearing or shapepreservation of the transplant are needed. Transplantation of a mixtureof both DBM and MBM together with BMC should enable the best of theadvantages of both: (a) significantly prolonging the period of osteo-and chondrogenic activity (with DBM acting fast and MBM after a delay);(b) improving the shape preservation of the implant throughout the wholeperiod of new tissue formation.

[0219] Addition of BMP to the mixture of BMC and DBM has considerablyaccelerated formation of new tissue both in the osteochondral complex ofthe knee joint and in the flat bones of the skull.

1. Use of a mixture comprising bone marrow cells (BMC) and demineralizedbone matrix (DBM) and/or mineralized bone matrix (MBM) as a graft ofmesenchymal progenitor cells for transplantation into a joint and/or acranio-facial-maxillary bone of a subject in need.
 2. The use accordingto claim 1, wherein said transplantation is for restoring and/orenhancing the formation of a new hyaline cartilage and subchondral bonestructure.
 3. The use according to any one of claims 1 or 2, whereinsaid transplantation is for generation of new osteochondral complexconsisting of articular cartilage and subchondral bone.
 4. The useaccording to any one of claims 1-3, wherein said BMC are allogeneic orsaid subject's own.
 5. The use according to any one of claims 1 to 4,further comprising active agents, preferably selected from bonemorphogenetic proteins (BMPs), immunosuppressants, immunomodulators,antibiotics and anti-inflammatory agents.
 6. The use according to anyone of claims 1-5, wherein said subject is a mammal.
 7. The useaccording to any one of claims 1 to 6, wherein the DBM and/or MBM are ofvertebrate origin.
 8. The use according to any one of claims 1 to 7,wherein the DBM and/or MBM are of human origin.
 9. The use according toany one of claims 1 to 8, wherein the DBM or MBM are in powder or sliceform.
 10. The use according to any one of claims 1 to 9, wherein theparticle size of the DBM is about 50 to 2500μ.
 11. The use according toclaim 10, wherein the particle size of the DBM is about 250 to 500μ. 12.The use according to any one of the claims 1 to 11, wherein the ratiobetween BMC and DBM is between 1:1 and 20:1 (volume:volume).
 13. The useaccording to claim 12, wherein the ratio between BMC and DBM is between2:1 and 9:1 (volume:volume).
 14. The use according to claim 13, whereinthe ratio between BMC and DBM is 4:1 (volume:volume).
 15. The useaccording to any one of claims 1 to 14, wherein said mammal is a human.16. The use according to any one of the preceding claims, wherein saidtransplantation is for the treatment of a patient suffering from any oneof hereditary or acquired bone disorder, hereditary or acquiredcartilage disorder, a malignant bone or cartilage disorder, metabolicbone diseases, bone infections, conditions involving bone or cartilagedeformities and Paget's disease.
 17. The use according to any one ofclaims 1 to 16, wherein said transplantation is for the treatment of apatient in need of any one of correction of complex fractures, bonereplacement and formation of new bone in plastic or sexual surgery. 18.The use according to any one of claims 13 to 17, wherein the number ofbone marrow cells in the mixture is from about 10⁶ to 10⁸ cells/ml. 19.The use according to any one of the preceding claims, further optionallycomprising a pharmaceutically acceptable carrier or diluent.
 20. The useaccording to any one of claims 1 to 19, further comprising additionalactive agents.
 21. Use of a mixture comprising BMC and DBM and/or MBM,in the preparation of a composition for the treatment of a damaged jointor degenerative arthropathy, and/or for the treatment of a patientsuffering from any one of hereditary or acquired bone disorder,hereditary or acquired cartilage disorder, a malignant bone or cartilagedisorder, metabolic bone diseases, bone infections, conditions involvingbone or cartilage deformities and Paget's disease.
 22. Use of a mixturecomprising BMC and DBM and/or MBM, in the preparation of a compositionto be used for transplantation, for restoring and/or enhancing theformation of a new hyaline cartilage and subchondral bone structure. 23.A method for transplantation of a mixture comprising BMC with DBM and/orMBM and optionally further comprising pharmaceutically acceptablecarrier or diluent, into a joint or a cranio-facial-maxillary bone of asubject in need, wherein said method comprises introducing into saidjoint or bone a mixture comprising BMC and DBM and/or MBM, or acomposition as prepared in claim
 21. 24. The method according to claim23, wherein said mixture is administered non-invasively by a syringe, anarthroscopic procedure or by open surgery into the site of implantation.25. The method according to claim 24, for support of implants of joints,cranio-facial-maxillary bones, or other musculoskeletal implants.
 26. Amethod of treating a damaged joint or degenerative arthropathyassociated with malformation and/or dysfunction of cartilage and/orsubchondral bone in a mammal in need of such treatment, comprisingadministering into an affected joint or bone of said mammal a mixturecomprising BMC with DBM and/or MBM, said mixture optionally furthercomprising a pharmaceutically acceptable carrier or diluent and/oradditional active agents.
 27. The method according to claim 26, whereinthe BMC are either allogeneic or said mammal's own.
 28. The methodaccording to any one of claims 26 or 27, wherein said DBM or MBM are ina slice, powder, gel, semi-solid or solid form embedded in orencapsulated in polymeric or biodegradable materials.
 29. The methodaccording to claim 28, wherein the particle size of said DBM is about 50to 2500μ.
 30. The method according to claim 29, wherein the particlesize of said DBM is about 250 to 500μ.
 31. The method according to anyone of the preceding claims, wherein the ratio between the transplantedBMC and DBM is between 1:1 and 20:1 (volume:volume).
 32. The methodaccording to claim 31, wherein the ratio between the transplanted BMCand DBM is between 2:1 and 9:1 (volume:volume).
 33. The method accordingto claim 32, wherein the ratio between the transplanted BMC and DBM is4:1 (volume:volume).
 34. A non-invasive implantation method for supportof implants of joints or other musculoskeletal implants, comprisingintroducing a graft into a joint or a cranio-facial-maxillary bone of asubject in need, wherein said graft comprises a mixture of BMC and DBMand/or MBM.
 35. A kit for performing transplantation into a joint or acranio-facial-maxillary bone of a mammal of a mixture as defined in anyone of claims 1-20, wherein said kit comprises: (a) the mixture asdefined in any one of claims 1-20; (b) a BM aspiration needle; (c) anintra-osseous bone drilling burr; (d) a needle with a thick lumen forinfusion of viscous bone marrow-DBM mixture; (e) a 2-way lumen connectorfor simultaneous mixing of BMC-DBM and diluent; (f) a medium formaintaining BMC; and optionally (g) cryogenic means for handling andmaintaining BMC or BMC together with DBM.
 36. The kit according to claim35, optionally further comprising a carrier and/or diluent for themixture.